The present invention relates to the production of hybrid seed in plants. In particular, the invention relates to a method of hybrid seed production using as one parent of the hybrid a plant transformed with a chimeric gene which when expressed in the female reproductive structures yields a protein that catalyzes the conversion of an exogenously-applied protoxin to a toxin, thereby rendering fertilization ineffective. Also included in the invention is the use of the conditional female-sterile plants in combination with conditional male-sterile plants to more efficiently produce hybrid seed, chimeric genes useful for the invention, transgenic plants comprising the chimeric genes, and novel promoters useful for expression in the female reproductive structures of a plant.
Heterosis in crop plants has received considerable attention because of its marked effect on yield improvement. The increased productivity on crossing different strains of maize was first noted in the late 19th century and was then developed according to systematic genetic procedures. The usual method for raising hybrid maize is to establish many inbred lines, make intercrosses, and determine which hybrids are more productive in a given locality.
The success of hybrid maize motivated plant breeders to explore the existence and magnitude of hybrid vigor in many other species with economic importance. In general, hybrids exhibit increase yields in comparison to non-hybrid varieties. Hybrids are usually more efficient in use of growth factors and give a greater return per unit for the growth factors such as water and fertilizer. Under stress hybrids are generally superior to parental cultivars, with a more stable performance over a wide range of environments. With hybrids, there is uniformity in product and maturity that often facilitates harvest and increases the value of the product in the marketplace. The hybrid may also combine characters that are difficult or impossible to combine in other ways. This is particularly true of many interspecific and intergeneric hybrids. The general conclusion from research is that hybrid vigor, a common phenomenon in plants, is of sufficient magnitude to warrant commercial exploitation if appropriate techniques can be devised.
Hybrid vigor has been recognized as a wide-spread phenomenon in plants and animals for many years. Commercial hybrids are now used extensively in many crops, including maize, sorghum, sugar beet, and sunflower. Other large acreage crops such as wheat, barley and rice are still primarily grown as inbred varieties. Research is being conducted on these and other crops that may permit the wide-spread use of commercial hybrids in the future, but the primary limiting factor in new hybrid crop development is the lack of economical hybrid seed production methods in these crops.
Traditionally, large-scale hybrid seed production is accomplished by planting separate rows or blocks of female parent lines and the male parent lines to pollinate them. Only the seed produced on the female parent rows is harvested. To ensure that this seed is hybrid seed uncontaminated with selfed seed, pollination control methods must be implemented on the female parent plants to ensure that seeds formed on them result from cross-pollination and not self-pollination. Known pollination control mechanisms are generally mechanical, chemical, or genetic.
Elimination of fertile pollen from the female parent can be achieved by hand emasculation in some species such as maize, a monoecious species. Such elimination of fertile pollen is achieved by pulling or cutting the male inflorescence (tassel) from plants in the female parent population. This simple procedure prevents self-fertilization by mechanically detasseling female plants before pollen shed to prevent selfing. However, most major crop plants of interest have functional male and female organs within the same flower making emasculation impractical. Even where practical, this form of hybrid seed production is extremely labor intensive and hence expensive. To eliminate the laborious detasseling that is necessary to prevent self-fertilization in hybrid crosses, genetic factors which produce male-sterility have been used in some species.
Male-sterility in the female parent can be controlled by nuclear genes or by a cytoplasmic-genetic system. Genetic male-sterility is controlled by nuclear genes in which the alleles for sterility generally are recessive to the alleles for fertility. Genetic male-sterility occurs in many species. Usually, it is controlled by a single recessive gene that must be homozygous to cause male-sterility. Breeders who use genetic male-sterility for hybrid seed production usually develop a phenotypically uniform female line that segregates 50% male-sterile and 50% male-fertile individuals. Seed for these lines is increased in isolation by harvesting seed from plants homozygous for the male-sterility gene that are pollinated by plants heterozygous for the male-sterility gene, and hence male-fertile. To produce commercial hybrid seed with genetic male-sterility, the 50 percent of male-fertile female plants must be rogued from the field as soon as their fertility can be identified. The labor associated with roguing fertile plants from female plants has greatly restricted the use of genetic male-sterility in producing hybrid seed. There are several problems associated with this system for producing commercial hybrid seed. First, it is not possible to eliminate fertile plants from the desired male-sterile plants in the female population. Genetic male-sterile plants must be maintained by mating them with male-fertile individuals. Half of the F1 plants from such a cross would be sterile, but the remaining plants would be fertile. Thus, the unwanted male-fertile plants in the female population may disseminate pollen and reduce the effectiveness of the desired male parent.
The successful use of cytoplasmic male-sterility for commercial hybrid seed production requires a stable male-sterile cytoplasm, an adequate pollen source, and an effective system of getting the pollen from the male parent to the male-sterile female. Also, the cytoplasmic-genetic system of male sterility requires three lines to produce a single crossed hybrid; the A line (male-sterile), B line (male-fertile maintainer), and R line (male-fertile with restorer genes). Three-way crosses produced with cytoplasmic-genetic male sterility involved maintenance and production of four lines, an A and B line of one inbred and male-fertile inbreds of the other two.
Furthermore, the southern maize blight caused by Helminthosporium maydis, Race T, which severely attacked all maize hybrids with cytoplasmic male-sterile T cytoplasm, demonstrated the vulnerability of a hybrid seed production industry based on a single source of male-sterile cytoplasm. For hybrid maize, most seed producers have returned to hand or mechanical emasculation and wind pollination.
Hybrid seed may also be produced by the use of chemicals that block or kill viable pollen formation. These chemicals, called gametocides, are used to impart a transitory male-sterility. However, the expense and availability of the chemicals and the reliability of the applications limits the production of hybrid seed through the use of gametocides.
Molecular methods for hybrid seed production have also been described. Such methods transform plants with constructs containing anti-sense DNA and other genes which are capable of controlling the production of fertile pollen into plants. Such regenerated plants are functionally male-sterile and are used for the production of hybrid seed by crossing with pollen from male-fertile plants. The primary deficiencies of these approaches stem from the fact that the genetically engineered male sterility gene, whether it is an anti-sense or RNAse, can only be maintained in a heterozygous state. They are fundamentally the same as natural genetic male steriles in that they must be maintained by crossing to isogenic male fertile lines. This is most problematic in the hybrid cross field where the acreage is large and yield is critical. The heterozygous female parent, of which only 50% will be male sterile, must be planted in rows next to the pollen donor male parent and the 50% fertile female parents removed. This is rendered easier in genetically engineered genetic male steriles because a herbicide resistance gene can be linked to the male sterility gene, and herbicide spray can be used to remove the fertile plants, but it still means that the female parent rows must be planted at double density in order to get the same yield per acre of our system. This will cause some yield loss due to competition. The herbicide spray also means yield loss because the resistant plants are never 100% immune to the herbicide, and the costs of spraying the chemical are considerable.
A shortcoming of these traditional hybrid seed production systems is the need to plant separate rows or blocks of the male and female parent lines. The physical distance between the male pollen donor and female pollen recipient plants results in less efficient pollen transfer, poor seed set on the female parent, the need to dedicate more production land to pollen donor plants, and less yield of hybrid seed per unit area of land. This shortcoming is especially acute in crop species such as wheat that release small amounts of pollen, and the pollen is not effectively carried by the wind. Traditional hybrid seed production methods when applied to wheat have required from one third to two thirds of the production field be dedicated to male pollen donor plants (Johnson and Schmidt, Adv. Agronomy 20:199-233 (1968); Wilson, Plant Breeding Reviews 303-309 (1989)). The result is the cost of hybrid wheat seed production is too high to sustain an industry despite the availability of hybrid seed production techniques and proven heterosis.
To achieve a more economical hybrid seed production system for wheat and other crops, it is necessary to move the male and female parent plants closer together to effect more efficient pollen transfer. Rather than being in separate blocks of rows so that seed from only the female parent plants can be harvested, the male and female parent plants need to be interplanted in the same rows meaning that the plants are centimeters, rather than meters apart. Since it would be impractical to harvest seed from only the female parents when so closely spaced to make parents, it is necessary to prevent formation of viable seed on the male parent plants in addition to preventing formation of viable pollen on the female parent plants.
One method of preventing formation of viable seed is the use of female-sterile plants. Naturally occurring female sterility has been reported in several crops (Honma and Phatak, Journal of Heredity 55:143-145 (1964); Sniezdo and Winiarczyk, Protoplasma 187:31-38 (1995); Justus and Meyer, Journal of Heredity 54:167-168 (1963); Hanna and Powell, Journal of Heredity 65:247-249 (1974); Brown and Bingham, Crop Science 24:1207-1208 (1984)) but there are problems in maintaining these lines and they are not used commercially. A method for constructing a dominant, female-sterility gene has been described (EP 412,006 A1 (1990); Goldman et al., EMBO Journal 13:2976-2984 (1994)), but again the maintenance of a female-sterile line containing this gene is problematic due to the inability to create a line homozygous for the female-sterility gene. A method for maintenance and use in hybrid seed production of this female-sterility gene has been described (EP 402,270 (1990)). However, it requires the introduction of a female-sterility gene, a restorer gene of a first male-sterility gene, a second male-sterility gene and two herbicide resistance genes in a complex series of sequential transformations to create the female-sterile male parent line, and it requires the introduction of the first male-sterility gene, a restorer gene of the female-sterility gene and an herbicide resistance gene in a complex series of sequential transformations to create the male-sterile female parent line. Herbicide treatment is needed to select the correct genotypes at each round of line multiplication, and to produce the hybrid seed the production field needs to be treated with one of the herbicides to kill off undesirable genotypes that are a result of the process. Although the above system could provide the economic advantage of interplanting of the male and female lines, it is too complex for commercial utility.
Accordingly, there is a need for a simple, economical method for hybrid seed production.
The present invention provides a method for hybrid seed production comprising producing a conditional female sterile plant comprising a female-preferential promoter operably linked to a coding sequence which encodes an enzyme which catalyzes the conversion of a protoxin to a toxin, interplanting the conditional female sterile plant with a male sterile plant, inducing female sterility by applying the protoxin to the conditional female sterile plant, and producing hybrid seed. In one preferred embodiment of the invention, the plant is either normally self-pollinated or normally cross-pollinated. In particularly preferred embodiments, the plant is selected from the group consisting of maize, wheat, and barley. Also provided by the invention are hybrid seeds produced by the method.
The invention further provides an expression cassette comprising a female-preferential promoter operably linked to a coding sequence which encodes an enzyme which catalyzes the conversion of a protoxin to a toxin. A preferred embodiment comprises a female-preferential promoter operably linked to the coding sequence of the argE gene. Preferred embodiments of female-preferential promoters consist of the promoter from either the B200i4-2 clone, the P26 clone or the P19 clone. A particularly preferred embodiment comprises the female-preferential promoter from either the B200i4-2 clone or the P19 clone operably linked to the argE coding sequence. Additional embodiments of coding sequences useful in the invention are those obtained from the P450sul monoxygenase gene, and the pehA gene.
Also provided by the invention are plants comprising the expression cassette comprising a female-preferential promoter operably linked to a coding sequence which encodes an enzyme which catalyzes the conversion of a protoxin to a toxin, and seeds of such plants.
Another object of the invention is the use of a protoxin in a method of inducing female fertility in a plant which comprises a female-preferential promoter operably linked to a coding sequence which encodes an enzyme which catalyzes the conversion of a protoxin to a toxin, and inducing female sterility by applying the protoxin to the plant.
Yet another object of the invention is the use of the coding sequence from the argE gene in a method for producing for hybrid seed where the argE coding sequence is operably linked to a female-preferential promoter which when expressed catalyzes the conversion of a protoxin to a toxin thereby inducing female sterility.